Epidermal growth factor receptor variant type III markedly accelerates angiogenesis and tumor growth via inducing c-myc mediated angiopoietin-like 4 expression in malignant glioma
© Katanasaka et al.; licensee BioMed Central Ltd. 2013
Received: 12 January 2013
Accepted: 17 April 2013
Published: 25 April 2013
Expression of the constitutively activated mutant EGFR variant III (EGFRvIII), the most common mutation in glioblastoma multiforme (GBMs), has been clinically correlated with tumor proliferation, invasion, and angiogenesis. In this study, we examined the role of EGFRvIII on the tumor microenvironment, especially on angiogenesis.
To study the role of EGFRvIII in tumor angiogenesis, we prepared LN229 glioblastoma transfected with enhanced green fluorescent protein (EGFP), wild-type EGFR, or EGFRvIII (LN229-WT or -vIII), and examined tumor growth and microvessel density in the tumors. Additionally, the potential angiogenic factors were identified by real-time PCR analysis, and the functions in LN229-vIII cells were examined.
LN229-vIII cells showed more aggressive tumor growth and higher vascularity as compared to LN229-WT cells in vivo, although there was no significant difference in the cell growth rates in vitro. We next investigated the expression of 60 angiogenesis-related factors to clarify the mechanisms underlying the difference in vascularity between tumor xenografts of LN229-vIII and LN229-WT. We found that the mRNA and protein expressions of angiopoietin-like 4 (Angptl4), a secreted protein involved in angiogenesis and metabolism regulation, were significantly induced by EGFRvIII overexpression, both in vitro and in vivo. Constitutive knockdown of Angptl4 in LN229-vIII using shRNA significantly decreased the microvessel density in the tumor xenografts and suppressed tumor growth. To clarify the regulatory mechanisms of Angptl4 by EGFRvIII, we analyzed the signaling pathways and transcription factors by pharmacological inhibition and RNA interference. U0126, an ERK signal inhibitor dramatically suppressed Angptl4 expression. The transcription factor c-Myc, which is regulated by ERK, was activated in the LN229-vIII cells and knockdown of c-Myc using siRNA also attenuated Angptl4 expression in the LN229-vIII cells. Furthermore, chromatin immunoprecipitation (ChIP) assay revealed increased recruitment of c-Myc to the promoter region of Angptl4 in the LN229-vIII cells.
In summary, we demonstrated that EGFRvIII induces Angptl4 expression through the ERK/c-Myc pathway and promotes tumor angiogenesis in malignant gliomas.
Glioblastoma multiforme (GBM), classified as a grade IV astrocytoma, has an extremely poor prognosis . Long-term survival of patients with malignant gliomas has not improved substantially despite the development of multimodality treatments, including cytoreductive surgery, adjuvant radiation therapy, and cytotoxic chemotherapy. In order to develop additional therapeutic strategies, further understanding of the molecular genetics, biology and immunology of gliomas is desired.
GBMs are distinguished pathologically from lower-grade anaplastic astrocytomas by the presence of necrosis and microvascular hyperplasia, a florid form of angiogenesis . Above all, a striking feature of GBMs is the presence of increasing neovascularization . Many studies have demonstrated that glioma growth is dependent on the generation of tumor-associated blood vessels [4, 5], therefore, use of antiangiogenic strategies is considered as a promising approach for the treatment of malignant gliomas.
There has been important progress in the elucidation of the molecular pathogenesis of malignant gliomas. Two common and highly specific genetic events associated with the GBM histology are epidermal growth factor receptor (EGFR) amplification and loss of the phosphatase and tensin homologue on chromosome 10 (PTEN) [6, 7]. Many studies have revealed that EGFR is functionally dysregulated in various tumors. Dysregulation of signal transduction processes affects a variety of downstream biological processes associated with gene transcription and protein translation, cell proliferation, migration, adhesion, invasion, and angiogenesis . Abnormalities of EGFR signaling have also been reported to be observed frequently in GBMs . EGFR gene amplification or overexpression is detected in approximately 40% of patients with these tumors [10, 11].
The EGFR variant type III (EGFRvIII), the most common mutation of EGFR in GBMs, is reported to be present in 25% to 33% of all cases of GBMs, but only in those showing EGFR amplification and overexpression . EGFRvIII overexpression has been shown to induce tumor growth of GBMs  and reported to be correlated with a poor prognosis in clinical settings [14, 15]. This EGFR variant is the result of deletion of exons 2 to 7 including the extracellular ligand-binding domain, and its receptor tyrosine kinase is constitutively active . Because it is not present in normal tissues, it is considered as a potential target for tumor-specific therapy. Currently, considerable effort is being made for the development of anti-EGFRvIII agents, such as vaccines and specific antibodies [7, 16, 17].
EGFR signaling promotes not only cell growth, but also angiogenesis by induction of proangiogenic factors such as the vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) . Although the NF-kB/IL-8 pathway contributes to tumor angiogenesis in EGFRvIII-overexpressing glioblastomas , the EGFRvIII signaling pathways involved in the promotion of angiogenesis have not yet been clearly elucidated. In this study, we show the involvement of EGFRvIII in tumor angiogenesis in LN229, a GBM cell line, and that the induction of angiopoietin-like 4 (Angptl4) expression by c-Myc is involved in EGFRvIII-induced angiogenesis.
Promotion of tumor angiogenesis by EGFRvIII overexpression
Real-time PCR analysis for identification of EGFRvIII-related angiogenic factors
Knockdown of Angptl4 suppressed the growth of EGFRvIII-overexpressing tumors and tumor angiogenesis
Transcriptional regulation of Angptl4 by c-Myc
Although EGFRvIII has been shown to promote tumor growth of gliomas through various signaling pathways , the key signal molecules involved in the alteration of the tumor microenvironment have not yet been fully elucidated. In this study, we investigated whether EGFRvIII contributes to tumor angiogenesis, and showed dramatic increases in the microvessel density and vascular permeability in tumor xenografts of LN229-vIII as compared to LN229-WT in mice, consistent with the results of a previous study . Considering that hypervascularity is a distinctive pathological characteristic of malignant gliomas [31, 32], the EGFRvIII expression status may have a great impact on the clinical picture. Although EGFR is known to promote angiogenesis by induction of proangiogenic factors, such as VEGF-A and interleukin 8 [18, 33], no dramatic induction of angiogenesis by wtEGFR was observed in our experiments. This difference leads to the speculation that constitutive activation of EGFR may trigger striking induction of various transcripts, including pro-angiogenic factors. In order to examine the molecular mechanisms underlying the induction of angiogenesis by EGFRvIII, the expressions of 60 angiogenic factors in LN229 cells were examined by real-time PCR analysis. Although VEGF-A is a representative angiogenic factor and a possible therapeutic target for glioblastoma , VEGF-A induction by EGFRvIII was observed only to a certain extent in vivo (data not shown), and not at all in vitro (Additional file 3: Table S1). Among the 60 angiogenic factors, we first found that Angptl4 expression was significantly induced by EGFRvIII overexpression, and that Angptl4 acts as a pro-angiogenic factor in tumor xenografts. Recently, Bonavia, et al. showed that the NF-kB/IL-8 pathway plays important roles in EGFRvIII-induced angiogenesis and growth in gliomas , however, no significant change of the IL-8 expression was observed in our in vitro experiment (Additional file 3: Table S1). It is likely that the differences between our results and those of the previous report are related to differences in the cell lines.
The molecular mechanisms of Angptl4-induced angiogenesis in malignant gliomas still remain largely unknown. Angptl4 is expressed in the liver, adipose tissue and placenta, as also in ischemic tissues . It is a member of the angiopoietin family and is a target of members of the peroxisome proliferator-activated receptor (PPAR) family, which are known as metabolic-response transcription factors . It has been reported that expression of Angptl4 is upregulated under various conditions including hypoxia and caloric restriction, and transcription factors such as PPARγ and Smad have been shown to regulate its expression [35, 37, 38]. Increased Angptl4 expression has been shown in a variety of tumor tissues, such as oral Kaposi’s sarcoma, esophageal squamous cell carcinoma, gastric cancer, and colorectal cancer [39–42]. Since a number of reports have indicated the effects of Angptl4 on angiogenesis, including endothelial cell proliferation, migration, differentiation, endothelial cell adhesion, and vascular permeability [43–46], it seems likely that Angptl4 contributes to the increased angiogenesis and vascular permeability in gliomas formed by EGFRvIII cells. Moreover, it has been demonstrated that Angptl4 disrupts vascular endothelial cell-cell junctions and promotes lung metastasis of breast cancer cells expressing transforming growth factor-β , while preventing metastasis of melanoma cells  and also inhibiting angiogenesis . These diverse and often conflicting results suggest that Angptl4 exhibit tissue-specific activity and act in accordance with the prevailing cellular environment.
Our results suggest that Angptl4 transcription is regulated, at least partially, by EGFRvIII/ERK/c-Myc-mediated signaling. EGFR activation induces Ras/MEK/ERK phosphorylation, and phosphorylated ERK activates various transcription factors. It has been shown that MAPK signaling contributes to Angptl4 expression . Myc is known as an ERK-activated transcription factor . Wild-type EGFR expression, as compared to mock, increased tumor growth and Angptl4 expression in vivo, and also activated ERK phosphorylation in the LN229 cells; however, the degree of activation was not significantly different from that induced by EGFRvIII expression (data not shown). These data suggest that, although the MAPK pathway plays an important role in c-Myc activation, other factors are also involved in the marked activation of c-Myc and induction of Angptl4 expression in the LN229-vIII cells. The promoter region of Angptl4 contains the consensus sequence of c-Myc, ‘CACGTG’. The results of the ChIP assay revealed enhanced binding between c-Myc and the promoter region of Angptl4 in LN229-vIII cells, suggesting that the transcriptional regulation of Angptl4 by c-Myc might contribute to the induction of angiogenesis in gliomas. An MEK inhibitor was also found to markedly inhibit Angptl4 expression in EGFRvIII-overexpressing LN229 cells. In a previously reported study, combined use of an MEK inhibitor with a PI3K inhibitor effectively suppressed the growth of gliomas . MEK inhibitors have been examined in clinical trials for various cancers, and their potential usefulness in the treatment of gliomas has been suggested.
In conclusion, we demonstrated in this study that EGFRvIII induces Angptl4 expression through the ERK/c-Myc pathway, and that Angptl4 is a possible inducer of tumor angiogenesis in gliomas expressing EGFRvIII. Since EGFRvIII strongly induces neovascularization in the tumors, expression of EGFRvIII or Angptl4 may be a possible biomarker for predicting the effectiveness of antiangiogenic therapy, as well as serve as a therapeutic target, although further studies are needed.
The human glioblastoma cell lines LN229 (American Tissue Culture Collection) were maintained in Dulbecco’s minimal essential medium (DMEM, Sigma) supplemented with streptomycin (100 μg/ml), penicillin (100 units/mL), and 10% heat-inactivated fetal bovine serum (FBS) at 37°C under 5% CO2 in a humidified chamber. The cDNA for wild-type EGFR or EGFRvIII was transfected into LN229 cells by a retrovirus vector, as described previously , and the transfected cells were selected by GFP expression from the viral expression vector using a cell sorter (BD Biosciences).
Cell proliferation assay
LN229 cells (1,000 cells/well) were seeded into a 96-well microtiter plate. After incubation for 24-96 h at 37ºC, the cell viability was measured with a Cell Counting Kit-8 (Dojindo, Tokyo, Japan) in accordance with the manufacturer’s instructions.
RNA isolation, reverse-transcription PCR, and real-time PCR
Total RNA was isolated using Isogen (Nippon Gene, Tokyo, Japan) and the resulting RNA was reverse-transcribed with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Tokyo, Japan). Real-time PCR assay was performed on a StepOnePlus (Applied Biosystems) using the TaqMan Gene Expression Assays or a TaqMan Array Gene Signature 96-Well Plate (Angiogenesis, human, Applied Biosystems). The relative real-time PCR quantification was based on a comparative quantitation method.
Western blotting was performed as described previously , with some modifications. The cells were washed with ice-cold PBS and lysed with M-PER (PIERCE, Tokyo, Japan) containing protease and phosphatase inhibitors. The protein concentration was determined using a BCA protein assay kit (PIERCE). The protein samples were mixed with SDS-PAGE sample buffer (2% SDS, 10% glycerol, 6% 2-mercaptoethanol, 50 mM Tris-HCl; pH 6.8), and an equal amount of proteins in each sample was subjected to SDS-PAGE. The separated proteins were transferred to a PVDF membrane (Millipore, Bedford, MA) and blocked with 5% skim-milk in TBST (0.9% NaCl, 0.1% Tween20, 20 mM Tris-HCl; pH7.4). The primary antibodies used were anti-EGFR antibody (BD Pharmingen, NJ) and anti-actin antibody (Sigma). Horseradish peroxidase (HRP)-conjugated antibodies (Cell Signaling Technology, Tokyo, Japan) were used as the secondary antibodies. The PVDF membrane was developed with the ECL reagent (GE Healthcare, Buckinghamshire, UK).
Tumor xenograft model
LN229 cells were subcutaneously implanted (3.0 × 106 cells/mouse) into the posterior flanks of 4-week old female BALB/c nu/nu mice. The tumor sizes were monitored as described previously . Animal studies were carried out according to the Guideline for Animal Experiments, drawn up by the Committee for Ethics in Animal Experimentation of the National Cancer Center, which meet the ethical standards required by law and the guidelines about experimental animals in Japan.
Microvessel density analysis
After tumor implantation, the mice were sacrificed under diethyl ether anesthesia, and the tumors were dissected and weighed. Immunostaining was performed as described previously . The tumor tissues were embedded and frozen with dry ice/ethanol. Tumor frozen sections (7 μm) were prepared and air-dried for at least 1 h. The sections were fixed with cold acetone, blocked in goat serum for 10 min at room temperature, and then incubated with anti-mouse CD31 rat monoclonal antibody (BD Pharmingen) for 18 h at 4°C. The sections were then stained with ABC Elute kit, or anti-rat IgG-Alexa fluor 555 conjugates (Molecular Probes, Inc.) for immunohistochemistry and immunofluorescent staining, respectively. After mounting the sections, the images were examined and scanned with Biozero (Keyence, Tokyo, Japan) at 20 × magnification. For quantitative analysis, the vascular area/mm2 in the tumors was quantified by counting the CD31-positive area in independent hotspots of at least four different microscopic fields in each of five mice/group, using the ImageJ software. The four fields were averaged in each tumor and the averages for each animal used to express the final count ± SEM.
The in vivo vascular permeability assay was performed as described previously with some modifications . The tumor-implanted mice were intravenously injected with TexasRed conjugated dextran (50 mg/kg/mouse, Mw 70,000, Molecular probes, Inc). At 6 h after the injection, Alexa647-conjugated Isolectin IB4 (Molecular probes, Inc) was injected for fluorescent staining of the blood vessels. After 10 minutes, perfusion fixation was performed under ether anesthesia and the tumors were extracted from the mice. The extracted tumors were frozen and sectioned as described above. The sections were fixed with 4% paraformaldehyde, mounted, and observed by fluorescent microscopy as described above.
Enzyme-linked immunosorbent assay (ELISA)
LN229 cells were seeded (3.0 × 105 cells) in a 35-mm dish and incubated overnight. The medium was refreshed and the culture dish was incubated for a further 48 h at 37°C. The culture medium was collected and centrifuged at 1,000 g for 10 min. The supernatant was recovered and ELISA for Angptl4 was performed using the Human Angiopoietin-like 4 DuoSet ELISA kit (R&D Systems, Minneapolis, MN) with a sensitivity of 1.25 ng⁄mL, an intra-assay coefficient of variation of 0.6–7.6%, and an inter-assay coefficient of variation of 8.5–11.2%. The assay was performed in accordance with the manufacturer’s instructions. The remaining cells on the dishes were lysed and the amount of protein was measured by a BCA protein assay. Tumor tissues extracted from the mice were homogenized in PBS (–) and centrifuged at 10,000 × g for 10 min at 4°C. The supernatant was collected and ELISA was performed as described above. Duplicate measurements were performed in a single experiment.
Electrophoretic mobility shift assay (EMSA)
Nuclear fractions were extracted from the LN229 cells using a Nuclear Extraction kit (Panomics, Redwood City, CA). The EMSA binding assay was carried out using a Panomics EMSA “gel shift” kit in accordance with the manufacturer’s instructions. Assays were conducted using a biotin-labeled double-stranded oligonucleotide having a consensus recognition sequence for Myc/Max purchased from Panomics. Protein-DNA complexes were separated using nondenaturing PAGE. The oligonucleotides were secondarily probed with HRP-conjugated streptavidin and developed with the component solution by LAS4000.
The RNAi experiment was performed with the Lipofectamine RNAiMAX reagent (Invitrogen, Tokyo, Japan) in accordance with the manufacturer’s instructions. The sequences of siRNA for c-Myc were 5′-AGA CCU UCA UCA AAA ACA UTT-3′ (sense) and 5′-AUG UUU UUG AUG AAG GUC UCG-3′ (antisense), which were designed by Ambion, and the non-silencing control siRNA was purchased from Invitrogen. After incubation with the siRNA for 48 h at 37°C, the mRNA expressions of c-Myc and Angptl4 were quantitatively determined by real-time PCR. Short hairpin RNA targeting the Angptl4-including entry vector was designed and prepared by Invitrogen. The shRNA was subcloned to a retrovirus vector and used in the experiments as described in a previous study .
The ChIP assay was performed using the ChIP IT Express kit (Active Motif, Carlsbad, CA), in accordance with the manufacturer’s instructions. LN229 cells were fixed with 1% formaldehyde for 10 min. The cells were then washed, lysed, and sonicated to reduce DNA lengths to the range of 200 to 1500 bp. The chromatin/DNA complexes were incubated with antibodies to c-Myc (Santa Cruz Biotechnology, CA) or IgG (Cell Signaling Technology) overnight at 4°C. The immune complexes were precipitated, eluted, reverse-crosslinked, and treated with proteinase K. After extraction of the DNA fragments, real-time PCR analysis was performed using Power SYBR green PCR master mixes (Applied Biosystems). The primer for the promoter of Angptl4 was purchased from BioScience (Fredrick, MD), and was as follows: forward, 5′-TAC TAG CGG TTT TAC GGG CG-3′; reverse, 5′-TCG AAC AGG AGG AGC AGA GAG CGA-3′. The predicted PCR product included a c-Myc binding sequence. Relative enrichment was comparatively calculated using IgG negative control as described in eBioScience instructions.
Significant differences were analyzed by an unpaired Student’s t-test or analysis of variance (ANOVA) with Tukey’s post-hoc test using the GraphPad Prism software (Version 5.0). p < 0.05 was considered to indicate statistically significant difference.
This study was supported in part by a Research Resident Fellowship from the Third Term Comprehensive 10-Year Strategy for Cancer Control and Health and Labor Sciences Grants, Research on Advanced Medical Technology.
- Furnari FB, Fenton T, Bachoo RM, Mukasa A, Stommel JM, Stegh A, Hahn WC, Ligon KL, Louis DN, Brennan C: Malignant astrocytic glioma: genetics, biology, and paths to treatment. Genes Dev. 2007, 21: 2683-2710. 10.1101/gad.1596707.View ArticlePubMedGoogle Scholar
- Rong Y, Belozerov VE, Tucker-Burden C, Chen G, Durden DL, Olson JJ, Van Meir EG, Mackman N, Brat DJ: Epidermal growth factor receptor and PTEN modulate tissue factor expression in glioblastoma through JunD/activator protein-1 transcriptional activity. Cancer Res. 2009, 69: 2540-2549. 10.1158/0008-5472.CAN-08-1547.PubMed CentralView ArticlePubMedGoogle Scholar
- Maxwell M, Naber SP, Wolfe HJ, Hedley-Whyte ET, Galanopoulos T, Neville-Golden J, Antoniades HN: Expression of angiogenic growth factor genes in primary human astrocytomas may contribute to their growth and progression. Cancer Res. 1991, 51: 1345-1351.PubMedGoogle Scholar
- Kreisl TN, Kim L, Moore K, Duic P, Royce C, Stroud I, Garren N, Mackey M, Butman JA, Camphausen K: Phase II trial of single-agent bevacizumab followed by bevacizumab plus irinotecan at tumor progression in recurrent glioblastoma. J Clin Oncol. 2009, 27: 740-745. 10.1200/JCO.2008.16.3055.PubMed CentralView ArticlePubMedGoogle Scholar
- Norden AD, Drappatz J, Wen PY: Antiangiogenic therapies for high-grade glioma. Nat Rev Neurol. 2009, 5: 610-620. 10.1038/nrneurol.2009.159.View ArticlePubMedGoogle Scholar
- Endersby R, Baker SJ: PTEN signaling in brain: neuropathology and tumorigenesis. Oncogene. 2008, 27: 5416-5430. 10.1038/onc.2008.239.View ArticlePubMedGoogle Scholar
- Sampson JH, Archer GE, Mitchell DA, Heimberger AB, Herndon JE, Lally-Goss D, McGehee-Norman S, Paolino A, Reardon DA, Friedman AH: An epidermal growth factor receptor variant III-targeted vaccine is safe and immunogenic in patients with glioblastoma multiforme. Mol Cancer Ther. 2009, 8: 2773-2779. 10.1158/1535-7163.MCT-09-0124.PubMed CentralView ArticlePubMedGoogle Scholar
- Nicholas MK, Lukas RV, Jafri NF, Faoro L, Salgia R: Epidermal growth factor receptor - mediated signal transduction in the development and therapy of gliomas. Clin Cancer Res. 2006, 12: 7261-7270. 10.1158/1078-0432.CCR-06-0874.View ArticlePubMedGoogle Scholar
- Huang PH, Xu AM, White FM: Oncogenic EGFR signaling networks in glioma. Sci Signal. 2009, 2: re6-10.1126/scisignal.287re6.PubMedGoogle Scholar
- Frederick L, Wang XY, Eley G, James CD: Diversity and frequency of epidermal growth factor receptor mutations in human glioblastomas. Cancer Res. 2000, 60: 1383-1387.PubMedGoogle Scholar
- Gan HK, Kaye AH, Luwor RB: The EGFRvIII variant in glioblastoma multiforme. J Clin Neurosci. 2009, 16: 748-754. 10.1016/j.jocn.2008.12.005.View ArticlePubMedGoogle Scholar
- Aldape KD, Ballman K, Furth A, Buckner JC, Giannini C, Burger PC, Scheithauer BW, Jenkins RB, James CD: Immunohistochemical detection of EGFRvIII in high malignancy grade astrocytomas and evaluation of prognostic significance. J Neuropathol Exp Neurol. 2004, 63: 700-707.PubMedGoogle Scholar
- Nishikawa R, Ji XD, Harmon RC, Lazar CS, Gill GN, Cavenee WK, Huang HJ: A mutant epidermal growth factor receptor common in human glioma confers enhanced tumorigenicity. Proc Natl Acad Sci USA. 1994, 91: 7727-7731. 10.1073/pnas.91.16.7727.PubMed CentralView ArticlePubMedGoogle Scholar
- Tang CK, Gong XQ, Moscatello DK, Wong AJ, Lippman ME: Epidermal growth factor receptor vIII enhances tumorigenicity in human breast cancer. Cancer Res. 2000, 60: 3081-3087.PubMedGoogle Scholar
- Heimberger AB, Hlatky R, Suki D, Yang D, Weinberg J, Gilbert M, Sawaya R, Aldape K: Prognostic effect of epidermal growth factor receptor and EGFRvIII in glioblastoma multiforme patients. Clin Cancer Res. 2005, 11: 1462-1466. 10.1158/1078-0432.CCR-04-1737.View ArticlePubMedGoogle Scholar
- Li D, Ji H, Zaghlul S, McNamara K, Liang MC, Shimamura T, Kubo S, Takahashi M, Chirieac LR, Padera RF: Therapeutic anti-EGFR antibody 806 generates responses in murine de novo EGFR mutant-dependent lung carcinomas. J Clin Invest. 2007, 117: 346-352. 10.1172/JCI30446.PubMed CentralView ArticlePubMedGoogle Scholar
- Fukai J, Nishio K, Itakura T, Koizumi F: Antitumor activity of cetuximab against malignant glioma cells overexpressing EGFR deletion mutant variant III. Cancer Sci. 2008, 99: 2062-2069.PubMedGoogle Scholar
- Hirata A, Ogawa S, Kometani T, Kuwano T, Naito S, Kuwano M, Ono M: ZD1839 (Iressa) induces antiangiogenic effects through inhibition of epidermal growth factor receptor tyrosine kinase. Cancer Res. 2002, 62: 2554-2560.PubMedGoogle Scholar
- Bonavia R, Inda MM, Vandenberg S, Cheng SY, Nagane M, Hadwiger P, Tan P, Sah DW, Cavenee WK, Furnari FB: EGFRvIII promotes glioma angiogenesis and growth through the NF-kappaB, interleukin-8 pathway. Oncogene. 2012, 31: 4054-4066. 10.1038/onc.2011.563.PubMed CentralView ArticlePubMedGoogle Scholar
- Estrada-Bernal A, Lawler SE, Nowicki MO, Ray Chaudhury A, Van Brocklyn JR: The role of sphingosine kinase-1 in EGFRvIII-regulated growth and survival of glioblastoma cells. J Neurooncol. 2011, 102: 353-366. 10.1007/s11060-010-0345-z.PubMed CentralView ArticlePubMedGoogle Scholar
- Kondo M, Asai T, Katanasaka Y, Sadzuka Y, Tsukada H, Ogino K, Taki T, Baba K, Oku N: Anti-neovascular therapy by liposomal drug targeted to membrane type-1 matrix metalloproteinase. Int J Cancer. 2004, 108: 301-306. 10.1002/ijc.11526.View ArticlePubMedGoogle Scholar
- Hoffmann J, Schirner M, Menrad A, Schneider MR: A highly sensitive model for quantification of in vivo tumor angiogenesis induced by alginate-encapsulated tumor cells. Cancer Res. 1997, 57: 3847-3851.PubMedGoogle Scholar
- Katanasaka Y, Ida T, Asai T, Shimizu K, Koizumi F, Maeda N, Baba K, Oku N: Antiangiogenic cancer therapy using tumor vasculature-targeted liposomes encapsulating 3-(3,5-dimethyl-1H-pyrrol-2-ylmethylene)-1,3-dihydro-indol-2-one, SU5416. Cancer Lett. 2008, 270: 260-268. 10.1016/j.canlet.2008.05.009.View ArticlePubMedGoogle Scholar
- Le Jan S, Amy C, Cazes A, Monnot C, Lamande N, Favier J, Philippe J, Sibony M, Gasc JM, Corvol P, Germain S: Angiopoietin-like 4 is a proangiogenic factor produced during ischemia and in conventional renal cell carcinoma. Am J Pathol. 2003, 162: 1521-1528. 10.1016/S0002-9440(10)64285-X.PubMed CentralView ArticlePubMedGoogle Scholar
- Stapleton CM, Joo JH, Kim YS, Liao G, Panettieri RA, Jetten AM: Induction of ANGPTL4 expression in human airway smooth muscle cells by PMA through activation of PKC and MAPK pathways. Exp Cell Res. 2010, 316: 507-516. 10.1016/j.yexcr.2009.12.004.PubMed CentralView ArticlePubMedGoogle Scholar
- Feldkamp MM, Lala P, Lau N, Roncari L, Guha A: Expression of activated epidermal growth factor receptors, Ras-guanosine triphosphate, and mitogen-activated protein kinase in human glioblastoma multiforme specimens. Neurosurgery. 1999, 45: 1442-1453. 10.1097/00006123-199912000-00034.View ArticlePubMedGoogle Scholar
- Shigematsu H, Gazdar AF: Somatic mutations of epidermal growth factor receptor signaling pathway in lung cancers. Int J Cancer. 2006, 118: 257-262. 10.1002/ijc.21496.View ArticlePubMedGoogle Scholar
- Ueda S, Basaki Y, Yoshie M, Ogawa K, Sakisaka S, Kuwano M, Ono M: PTEN/Akt signaling through epidermal growth factor receptor is prerequisite for angiogenesis by hepatocellular carcinoma cells that is susceptible to inhibition by gefitinib. Cancer Res. 2006, 66: 5346-5353. 10.1158/0008-5472.CAN-05-3684.View ArticlePubMedGoogle Scholar
- Gao SP, Mark KG, Leslie K, Pao W, Motoi N, Gerald WL, Travis WD, Bornmann W, Veach D, Clarkson B, Bromberg JF: Mutations in the EGFR kinase domain mediate STAT3 activation via IL-6 production in human lung adenocarcinomas. J Clin Invest. 2007, 117: 3846-3856. 10.1172/JCI31871.PubMed CentralView ArticlePubMedGoogle Scholar
- Meyer N, Penn LZ: Reflecting on 25 years with MYC. Nat Rev Cancer. 2008, 8: 976-990. 10.1038/nrc2231.View ArticlePubMedGoogle Scholar
- Maeda H, Wu J, Sawa T, Matsumura Y, Hori K: Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review. J Control Release. 2000, 65: 271-284. 10.1016/S0168-3659(99)00248-5.View ArticlePubMedGoogle Scholar
- Wen PY, Kesari S: Malignant gliomas in adults. N Engl J Med. 2008, 359: 492-507. 10.1056/NEJMra0708126.View ArticlePubMedGoogle Scholar
- Wu JL, Abe T, Inoue R, Fujiki M, Kobayashi H: IkappaBalphaM suppresses angiogenesis and tumorigenesis promoted by a constitutively active mutant EGFR in human glioma cells. Neurol Res. 2004, 26: 785-791. 10.1179/016164104225014139.View ArticlePubMedGoogle Scholar
- Jain RK, di Tomaso E, Duda DG, Loeffler JS, Sorensen AG, Batchelor TT: Angiogenesis in brain tumours. Nat Rev Neurosci. 2007, 8: 610-622.View ArticlePubMedGoogle Scholar
- Padua D, Zhang XH, Wang Q, Nadal C, Gerald WL, Gomis RR, Massague J: TGFbeta primes breast tumors for lung metastasis seeding through angiopoietin-like 4. Cell. 2008, 133: 66-77. 10.1016/j.cell.2008.01.046.PubMed CentralView ArticlePubMedGoogle Scholar
- Oike Y, Ito Y, Maekawa H, Morisada T, Kubota Y, Akao M, Urano T, Yasunaga K, Suda T: Angiopoietin-related growth factor (AGF) promotes angiogenesis. Blood. 2004, 103: 3760-3765. 10.1182/blood-2003-04-1272.View ArticlePubMedGoogle Scholar
- Veliceasa D, Schulze-Hoepfner FT, Volpert OV: PPARgamma and Agonists against Cancer: Rational Design of Complementation Treatments. PPAR Res. 2008, 2008: 945275-PubMed CentralView ArticlePubMedGoogle Scholar
- Tian L, Zhou J, Casimiro MC, Liang B, Ojeifo JO, Wang M, Hyslop T, Wang C, Pestell RG: Activating peroxisome proliferator-activated receptor gamma mutant promotes tumor growth in vivo by enhancing angiogenesis. Cancer Res. 2009, 69: 9236-9244. 10.1158/0008-5472.CAN-09-2067.PubMed CentralView ArticlePubMedGoogle Scholar
- Nakayama T, Hirakawa H, Shibata K, Abe K, Nagayasu T, Taguchi T: Expression of angiopoietin-like 4 in human gastric cancer: ANGPTL4 promotes venous invasion. Oncol Rep. 2010, 24: 599-606.View ArticlePubMedGoogle Scholar
- Shibata K, Nakayama T, Hirakawa H, Hidaka S, Nagayasu T: Clinicopathological significance of angiopoietin-like protein 4 expression in oesophageal squamous cell carcinoma. J Clin Pathol. 2010, 63: 1054-1058. 10.1136/jcp.2010.078600.View ArticlePubMedGoogle Scholar
- Hu J, Jham BC, Ma T, Friedman ER, Ferreira L, Wright JM, Accurso B, Allen CM, Basile JR, Montaner S: Angiopoietin-like 4: A novel molecular hallmark in oral Kaposi’s sarcoma. Oral Oncol. 2011, 47: 371-375. 10.1016/j.oraloncology.2011.02.018.PubMed CentralView ArticlePubMedGoogle Scholar
- Nakayama T, Hirakawa H, Shibata K, Nazneen A, Abe K, Nagayasu T, Taguchi T: Expression of angiopoietin-like 4 (ANGPTL4) in human colorectal cancer: ANGPTL4 promotes venous invasion and distant metastasis. Oncol Rep. 2011, 25: 929-935.View ArticlePubMedGoogle Scholar
- Kim I, Kim HG, Kim H, Kim HH, Park SK, Uhm CS, Lee ZH, Koh GY: Hepatic expression, synthesis and secretion of a novel fibrinogen/angiopoietin-related protein that prevents endothelial-cell apoptosis. Biochem J. 2000, 346: 603-610. 10.1042/0264-6021:3460603.PubMed CentralView ArticlePubMedGoogle Scholar
- Cazes A, Galaup A, Chomel C, Bignon M, Brechot N, Le Jan S, Weber H, Corvol P, Muller L, Germain S, Monnot C: Extracellular matrix-bound angiopoietin-like 4 inhibits endothelial cell adhesion, migration, and sprouting and alters actin cytoskeleton. Circ Res. 2006, 99: 1207-1215. 10.1161/01.RES.0000250758.63358.91.PubMed CentralView ArticlePubMedGoogle Scholar
- Ma T, Jham BC, Hu J, Friedman ER, Basile JR, Molinolo A, Sodhi A, Montaner S: Viral G protein-coupled receptor up-regulates Angiopoietin-like 4 promoting angiogenesis and vascular permeability in Kaposi’s sarcoma. Proc Natl Acad Sci USA. 2010, 107: 14363-14368. 10.1073/pnas.1001065107.PubMed CentralView ArticlePubMedGoogle Scholar
- Hermann LM, Pinkerton M, Jennings K, Yang L, Grom A, Sowders D, Kersten S, Witte DP, Hirsch R, Thornton S: Angiopoietin-like-4 is a potential angiogenic mediator in arthritis. Clin Immunol. 2005, 115: 93-101. 10.1016/j.clim.2004.12.002.View ArticlePubMedGoogle Scholar
- Galaup A, Cazes A, Le Jan S, Philippe J, Connault E, Le Coz E, Mekid H, Mir LM, Opolon P, Corvol P: Angiopoietin-like 4 prevents metastasis through inhibition of vascular permeability and tumor cell motility and invasiveness. Proc Natl Acad Sci USA. 2006, 103: 18721-18726. 10.1073/pnas.0609025103.PubMed CentralView ArticlePubMedGoogle Scholar
- Ito Y, Oike Y, Yasunaga K, Hamada K, Miyata K, Matsumoto S, Sugano S, Tanihara H, Masuho Y, Suda T: Inhibition of angiogenesis and vascular leakiness by angiopoietin-related protein 4. Cancer Res. 2003, 63: 6651-6657.PubMedGoogle Scholar
- Edwards LA, Verreault M, Thiessen B, Dragowska WH, Hu Y, Yeung JH, Dedhar S, Bally MB: Combined inhibition of the phosphatidylinositol 3-kinase/Akt and Ras/mitogen-activated protein kinase pathways results in synergistic effects in glioblastoma cells. Mol Cancer Ther. 2006, 5: 645-654. 10.1158/1535-7163.MCT-05-0099.View ArticlePubMedGoogle Scholar
- Fuse C, Ishida Y, Hikita T, Asai T, Oku N: Junctional adhesion molecule-C promotes metastatic potential of HT1080 human fibrosarcoma. J Biol Chem. 2007, 282: 8276-8283. 10.1074/jbc.M608836200.View ArticlePubMedGoogle Scholar
- Koizumi F, Shimoyama T, Taguchi F, Saijo N, Nishio K: Establishment of a human non-small cell lung cancer cell line resistant to gefitinib. Int J Cancer. 2005, 116: 36-44. 10.1002/ijc.20985.View ArticlePubMedGoogle Scholar
- Yonezawa S, Asai T, Oku N: Effective tumor regression by anti-neovascular therapy in hypovascular orthotopic pancreatic tumor model. J Control Release. 2007, 118: 303-309. 10.1016/j.jconrel.2006.12.024.View ArticlePubMedGoogle Scholar
- Fernandez-Lopez D, Faustino J, Daneman R, Zhou L, Lee SY, Derugin N, Wendland MF, Vexler ZS: Blood-brain barrier permeability is increased after acute adult stroke but not neonatal stroke in the rat. J Neurosci. 2012, 32: 9588-9600. 10.1523/JNEUROSCI.5977-11.2012.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.